1. Field of the Invention
The present invention relates to cellular manipulation devices and, more specifically, to a device that can perform poration, transfection, lysis and sorting of living cells.
2. Description of the Related Art
As reflected in the recent Proteomics special feature article (“Automated NanoElectrospray: A New Advance for Proteomics Researchers,” Laboratory News, 2002) Mass Spectrometry (MS) has become the technology of choice to meet today's unprecedented demand for accurate bioanalytical measurements, including protein identification. Although MS can be used to analyze biomolecules with very large molecular weights (up to several MegaDaltons (Mda)), these molecules must be first converted to gas-phase ions before they can be introduced into a mass spectrometer for analysis. Electrospray ionization (ESI) has proven to be an enormous breakthrough in structural biology because it provides a mechanism for transferring large biological molecules into the gas phase as intact charged ions. It is the creation of efficient conversion of a very small quantity of a liquid sample (proteins are very expensive and often very difficult to produce in sizable quantities) into gas-phase ions that is one of the main bottlenecks for using mass spectrometry in high throughput proteomics.
Conventional (micro and nano) capillary ESI sources, as well as the more recently developed MEMS-based electrospray devices, rely on application of strong electric field, which is used for focusing of the charged jet leading to jet tip instabilities and formation of small droplets of the analyte sample. As a result, the size and homogeneity of the formed droplets is determined by the magnitude and geometry of the applied electric field, thus requiring high voltages for generating sufficiently small micrometer or sub-micrometer droplets via the so-called Taylor cone nebulization. Reliance on the electrohydrodynamic Taylor cone focusing of the jet to form the mist of sufficiently small charged droplets leading to single ion formation imposes several fundamental and significant limitations on the capabilities of the conventional ESI interface.
One such problem is that a very large electric potential needs to be applied to the capillary tip (up to a few kilovolts relative to the ground electrode of the MS interface) to ensure formation of the stable Taylor cone, especially at higher flow rates and with poorly conducting organic solvents.
An additional problem is that the choice of suitable solvents is very much restricted to those featuring high electrical conductivity and sufficiently low surface tension. This restriction imposes severe limitations on the range of biological molecules that can be analyzed via ESI Mass Spectrometry. For example, use of pure water (the most natural environment for most biomolecules) as a solvent is difficult in conventional ESI since the required onset electrospray voltage is greater than that of the corona discharge, leading to an unstable Taylor cone, damage to the emitter and uncontrollable droplet/ion formation.
Since the conventional ESI relies on the disintegration of the continuous jet emanating from the Taylor cone into an aerosol of charged droplets, there is the limit to the lowest flow rate (and therefore the minimum sample size) that can be used during the analysis. For example, commercial products require the minimum sample volume to be about 3 μL.
Another problem is that sample utilization (i.e., fraction of the sample volume that is introduced and being used in MS analysis relative to the total volume of the electrosprayed sample) is very low due to uncontrollable nature of electrohydrodynamic atomization process that relies on the surface instabilities. Further, a significant dead volume (i.e., a fraction of the sample that cannot be pulled from the capillary by electrical forces) is unavoidable in any jet-based atomization process.
Still other problems are that commercially available ESI devices are very expensive because of the manufacturing difficulties, and limited usable lifetime because of the high voltage operation in a chemically-aggressive solvent environment.
An ability to extract DNA from or inject DNA into living cells is critical to any genetic, molecular biology, drug design and delivery, and pharmaceutical research and development work. Drug delivery, pharmaceutical, and biotech industries routinely need to be able to extract DNA from and inject DNA into a cell. This is probably the most critical step in many molecular biology and genetics modification protocols currently used.
Some methods of injecting DNA into cells involve poration of a group of cells. In poration, the cells are subjected to an energy field that causes pores in the cell membranes to dilate. Typically, many cells are placed in a field that varies spatially and those cells that are in the area of a certain field strength porate, while the rest do not. The low level of predictability and accuracy of poration results in a low yield and the inefficiency of requiring the technician to spend extra time sorting cells that have successfully porated from those that have not successfully porated.
Therefore, there is a need for a system for extracting and injecting materials into living cells with a high level of predictability and accuracy.